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At least 541 records · Page 30Linked to original sources

PubMed1989

A new neomycin phosphotransferase II solid phase assay in combination with polyacrylamide sodium dodecylsulphate gel electrophoresis.

A new general method for the determination of neomycin phosphotransferase (NPT) II (EC 2.7.1.95) activity in cell extracts after separation in SDS-polyacrylamide gels is described. The enzymatic activity of NPT II is restored after SDS-polyacrylamide gel electrophoresis by incubating the gel for 3 h (20 mM Tris-HCl buffer, pH 7.4). The enzymatic activity is determined by in situ phosphorylation of aminoglycoside antibiotics bound to solid supports and brought into direct contact with the gel surface. A novel, mechanically stable, negatively charged matrix was synthesized for use in this solid phase enzyme assay and compared to phosphocellulose and carboxymethylcellulose paper. This new method allows the easy and exact determination of the molecular weight of any fusion protein with NPT II by assaying the position of the enzymatic activity in the gel and a consecutive immunological reaction following protein transfer onto nitrocellulose membranes.

Aminoglycosides↗

PubMed1987

Quantitative assay of recombinant vaccinia virus-encoded neomycin phosphotransferase in infected eukaryotic cell lysates.

A method for the detection and quantitation of neomycin phosphotransferase (NPT II) activity in recombinant vaccinia virus (VV)-infected eukaryotic cell lysates is described. The assay is linear with respect to both protein concentration and time of incubation. Cytoplasmic extracts of cells infected with a recombinant VV expressing the bacterial neo gene exhibited NPT II levels more than 50-fold higher than those detected in extracts from either uninfected or VV-infected cells. These results indicate that interference from cellular or viral-induced ATPase activities is sufficiently low that NPT II enzyme activity can be measured in crude cell lysates without employing additional protein purification procedures.

Acetyltransferases↗

PubMed1993

Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene.

In Trypanosoma cruzi, the cause of Chagas' disease in Latin America, a large proportion of the antigenic proteins described to date have repetitive domains. In earlier work we identified a partial length cDNA, designated TCR27, encoding approx. 26 copies of a 14-amino acid repeat and a unique 61-amino acid C-terminal region. The goal of the current project was to replace the repetitive region of a TCR27 gene with the neomycin phosphotransferase gene (NEOr). A pBluescript-based vector was constructed in which the 0.9-kb NEOr coding region replaced the 2.9-kb internal repetitive segment of a TCR27 gene and was in frame with its nonrepetitive 5' coding sequence (pTCR27-2::NEO). Epimastigotes were electroporated in the presence of linearized pTCR27-2::NEO and transfected clones were selected on solid medium containing G418. Southern and Northern analyses of DNAs and RNAs from four G418-resistant clones showed that in all cases the repetitive region in the smaller of the two TCR27 genes (TCR27-2) had been replaced by NEOr. The absence of the native TCR27-2 protein in the transfected clones was confirmed by Western blot. In axenic cultures growth rates of epimastigotes bearing an interrupted TCR27-2 gene were not different from those of wild-type parasites. In addition, there was no relative impairment of the four transfected clones' ability to proliferate in cultured mammalian cells. The fact that the clones having the interrupted TCR27-2 gene were not impaired biologically suggests that the length of the repetitive region of the TCR27 protein is not a critical factor for survival.

Amino Acid Sequence↗

PubMed1986

Purification and characterization of ATP:AMP phosphotransferase from Mycobacterium marinum.

ATP:AMP phosphotransferase (EC 2.7.4.3) (adenylate kinase) has been purified 1746-fold from Mycobacterium marinum (ATCC 927) by successive column chromatography on DEAE-cellulose (DE-53), Reactive Blue agarose, Sephadex G-75, hydroxyapatite and, finally, DEAE-Sephadex A-50. The final enzyme preparation had a specific activity of 576 mumol/min per mg protein with an overall yield of 51%. The preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was estimated to have an Mr of 29500 and an isoelectric point of 6.7, properties which generally resemble those of the mitochondrial enzyme. Indeed, the two enzymes failed to separate when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The extinction coefficient (at 276 nm) was calculated to be 3.114 X 10(4) M-1 X cm-1 and E1%1cm = 10.556. Adenylate kinase was present at a concentration of 0.06 mg/g (wet weight) bacteria. Enzyme was stable for months in 60% glycerol in the freezer; at 4 degrees C, less than 5% of the activity was lost over a 7 day period.

Adenylate Kinase↗

PubMed1987

The beta-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation.

The beta-phosphoro[35S]thioate analogue of UDP-glucose ((beta-35S)UDP-Glc) is utilized with approximately the same efficiency as the parent compound by the UDP-glucose:glycoprotein glucose-1-phosphotransferase (glucosyltransferase), which catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose-containing oligosaccharides on acceptor glycoproteins. The same endogenous acceptor glycoproteins are labeled by the glucosyltransferase using [beta-32P]UDP-Glc and (beta-35S)UDP-Glc. However, in liver homogenates, incorporation from [beta-32P]UDP-Glc ceases to increase after about 4 min of incubation, while incorporation from (beta-35S)UDP-Glc persists for at least 1 h. This difference is due to an approx. 10-fold slower hydrolytic rate for the phosphorothioate analogue than for the parent compound, a finding similar to previous work showing that a variety of nucleases and phosphodiesterases are less efficient in cleaving phosphorothioate DNA than the native polymer.

Animals↗

PubMed1991

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci.

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Two open reading frames adjacent to the Escherichia coli K-12 transketolase (tkt) gene show high similarity to the mannitol phosphotransferase system enzymes from Escherichia coli and various gram-positive bacteria.

I have subcloned and sequenced the genes cmtB and cmtA of Escherichia coli K-12 which lie adjacent to the tkt gene on the chromosome. The genes cmtB and cmtA could encode a cytoplasmic protein (EIIA) and an integral-membrane protein (EIIBC), respectively, of the bacterial phospho enol pyruvate-dependent carbohydrate phosphotransferase system. High similarity to the Enzymes IIMtl of Escherichia coli K-12 (gene mtlA) and of Staphylococcus carnosus was detected, but the two genes did not complement mannitol-negative E. coli mutants without the use of a heterologous promoter.

Amino Acid Sequence↗

PubMed1993

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase and biosynthetic capacity during differentiation of hypocotyls of Vigna seedlings.

The relationship between the activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) and the capacity for biosynthesis of macromolecules was examined in segments from different parts of hypocotyls of etiolated seedlings of Vigna mungo and V. radiata. The relative ratio of the maximum activity of PFP to that of ATP-dependent phosphofructokinase (PFK) (PFP/PFK ratio) was high in young tissues and decreased with differentiation and ageing of the tissues. The highest level of fructose-2,6-bisphosphate was observed in the youngest part of hypocotyls of V. mungo. The level was markedly decreased with ageing of tissues. The levels of PPi and ATP were also higher in younger parts than in older parts of the hypocotyls, but the ratio of the level of PPi to that of ATP was almost constant in all parts of the hypocotyl. A good correlation was found between the PFP/PFK ratio and the biosynthetic capacity, as estimated from the rate of incorporation of [U-14C]sucrose into ethanol-insoluble macromolecules.

Aging↗

PubMed1987

Hydrogenosomal ATP:AMP phosphotransferase of Trichomonas vaginalis.

1. ATP:AMP phosphotransferase (adenylate kinase) is present in Trichomonas vaginalis, primarily with hydrogenosomal localization. 2. Adenylate kinase has been purified from hydrogenosome-enriched fractions by solubilization with Triton X-100 and KCl followed by affinity chromatography and gel filtration. 3. The enzyme has a Mr = 28,000, a broad pH optimum of pH 7-9, requirement for Mg2+ and specificity for adenine and deoxyadenine nucleotides. 4. The activity is competetively inhibited by P1,P5-di(adenosine-5') pentaphosphate (Ki 200 nM) and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoate). 5. Catalytic properties of this enzyme are similar to those of enzymes from other organisms. Other properties indicate its uniqueness, however, since its molecular mass and Ki for P1,P5-di(adenosine-5'-)-pentaphosphate bring it closer to the mitochrondrial isoenzyme, while it shares a requirement for reduced thiol groups with the cytosolic isoenzyme.

Adenylate Kinase↗

PubMed1992

Altered substrate specificity by substitutions at Tyr218 in bacterial aminoglycoside 3'-phosphotransferase-II.

Mutant aminoglycoside 3'-phosphotransferase II enzymes were produced in which Tyr218 was changed to serine, aspartic acid, or phenylalanine. In each case the mutation resulted in increased bacterial susceptibility to neomycin and kanamycin, while simultaneously increasing the Km values for these substrates. For the Ser and Asp mutants, bacterial resistance to amikacin increased, with a concomitant increase in affinity for this drug. Initial velocity studies indicated that the wild-type and mutant enzymes all followed Michaelis-Menten kinetics. Although these mutagenic substitutions changed the substrate specificity of these enzymes they did not alter the enzyme affinity for Mg(2+)-ATP.

Amino Acid Sequence↗

PubMed1993

Identification of Streptococcus mutans antigen D as the HPr component of the sugar-phosphotransferase transport system.

Extraction of Streptococcus mutans with the detergents HECAMEG and lauroyl sarcosinate preferentially extracted antigen D, previously identified as a low molecular mass wall-associated protein. Western blotting with specific antisera was used to demonstrate that this antigen is the HPr component of the sugar-phosphotransferase transport system. Non-denaturing electrophoresis indicated that HECAMEG selectively extracted only one of the two forms of HPr. It is suggested that this form of HPr may have a specific cell surface location.

Antigens, Bacterial↗

PubMed1983

Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae.

The plasmid-borne gene hph coding for hygromycin B phosphotransferase (HPH) in Escherichia coli has been identified and its nucleotide sequence determined. The hph gene is 1026 nucleotides long, coding for a protein with a predicted Mr of 39 000. The hph gene was placed in a shuttle plasmid vector, downstream from the promoter region of the cyc 1 gene of Saccharomyces cerevisiae, and an hph construction containing a single AUG in the 5' noncoding region allowed direct selection following transformation in yeast and in E. coli. Thus the hph gene can be used in cloning vectors for both pro- and eukaryotes.

Anti-Bacterial Agents↗

PubMed1984

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts.

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [gamma-32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

Animals↗

PubMed1986

Construction and uses of a new transposable element whose insertion is able to produce gene fusions with the neomycin-phosphotransferase-coding region of Tn903.

We describe the construction of a transposable element derived from the Mu phage that upon insertion is able to create a gene fusion between the region of Tn903 coding for neomycin phosphotransferase (NPT I), which confers resistance to aminoglycosides including kanamycin (KmR), neomycin and G418, and the control elements of the gene where the insertion occurs. A chloramphenicol (Cm) transacetylase gene (cat) that confers resistance to Cm is present in the transposon so that transposition events can be monitored even when no active fusions with the nptI coding region occur. The transposase gene is deleted and, therefore, this transposon is perfectly stable upon insertion. The properties of this new transposable element were studied by obtaining gene fusions between the Escherichia coli L-arabinose operon and 'nptI gene. In some of them the KmR phenotype is induced by arabinose. Insertions of this element in cloned fragments of the T-DNA region of Agrobacterium rhizogenes were also isolated. Some of them confer a KmR phenotype upon its E. coli carriers, which indicates that portions of the T-DNA are expressed in these cells.

Base Sequence↗

PubMed1988

A gene fusion system using the aminoglycoside 3'-phosphotransferase gene of the kanamycin-resistance transposon Tn903: use in the yeast Kluyveromyces lactis and Saccharomyces cerevisiae.

The aminoglycoside 3'-phosphotransferase type I (APHI)-coding gene of the bacterial transposon Tn903 confers resistance to kanamycin on bacteria and resistance to geneticin (G418) on many eukaryotes. We developed an APHI fusion system that can be used in the study of gene expression in these organisms, particularly in yeasts. The first 19 codons of the KmR (APHI) gene can be deleted, and replaced by other genes in a continuous reading frame, without loss of APH activity. Examples of vector constructions are given which are adapted to the yeast Kluyveromyces lactis transformation system. Their derivatives containing the 2 mu origin of replication can also be used in Saccharomyces cerevisiae.

Base Sequence↗

PubMed1988

Nucleotide sequences of the Escherichia coli nagE and nagB genes: the structural genes for the N-acetylglucosamine transport protein of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and for glucosamine-6-phosphate deaminase.

The genes coding for the enzymes of N-acetylglucosamine (GlcNAc) uptake and metabolism (nagA, nagB, and nagE) are located next to glutaminyl-tRNA synthetase gene (glnS) in the Escherichia coli genome. We determined the nucleotide sequence of the nagE (ptsN) gene, encoding the GlcNAc-specific enzyme II (NagE) of the phosphoenolpyruvate: sugar phosphotransferase system, and the sequence of the putative nagB gene, for glucosamine-6-phosphate deaminase. S1 mapping identified the mRNA transcript for nagE, indicating that nagE might be a sole constituent of the nagE operon, and divergent transcripts which are probably of the nagB, nagA genes. An evaluation of the hydrophobic and hydrophilic properties of NagE shows characteristics of a membrane protein. Also, NagE shows homologies to lactose permease and to the glucose-specific transport protein (enzyme IIGlc), and the glucose-specific phosphoryl carrier protein (enzyme IIIGlc). The latter two homologies are particularly interesting since no enzyme III-like protein for GlcNAc transport has been reported and enzyme IINag is of similar size as the combined enzymes IIGlc plus IIIGlc. This supports the idea that these two transport and phosphorylation systems may have evolved from a common ancestral gene.

Aldose-Ketose Isomerases↗

PubMed1989

A sensitive and simple assay for neomycin phosphotransferase II activity in transgenic tissue.

A new assay for the detection of the enzyme neomycin phosphotransferase II (NPTII) in crude cell extracts is described. The method is based on the chromatographic separation of the compounds resulting from the reaction of NPTII with kanamycin (Km) and [gamma-32P]ATP; the labelled Km.phosphate is subsequently detected by autoradiography. Chromatography is carried out on polyethyleneimine cellulose plates. This assay has been tested with bacterial, plant and animal crude extracts and comparisons with the assays in current use have been made. Our assay has several advantages: it is simple, rapid, sensitive and safe.

Adenosine Triphosphate↗

PubMed1990

Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon.

A series of isogenic strains harboring known deletions in the pts operon of Escherichia coli have been constructed by reverse genetics. Strains bearing deletions for the whole pts operon failed to grow on maltose or on carbon sources of the same class. In these strains the total cAMP synthesis was significantly lower than in a strain deleted only for the crr gene. This indicated that enzyme I or phosphorylated histidine-containing phosphotransferase protein in addition to its role in phosphorylating enzyme IIIGlc, is involved in adenylate cyclase (AC) activation or cAMP excretion. It was further shown that deletions in the pts operon do not affect synthesis of AC.

Adenylyl Cyclases↗